A wide variety of fermentation products can be made using sugars from lignocellulosic biomass as a substrate (Hahn-Hägerdal et al. 2006. Trends Biotechnol. 24:549-556; Jarboe et al. 2007 Adv. Biochem. Engin/Biotechnol. 108:237-261, Katahira et al. 2006 Appl. Microbiol. Biotechnol. 72:1136-1143, Tokiwa et al. 2008. Can. J. Chem. 86:548-555). Prior to fermentation, however, the carbohydrate polymers cellulose and hemicellulose must be converted to soluble sugars using a combination of chemical and enzymatic processes (Um et al. 2003 Appl Biochem Biotechnol. 105-108:115-125; Wyman et al. 2005. 96:2026-2032). Chemical processes are accompanied by side reactions that produce a mixture of minor products such as alcohols, acids, and aldehydes that have a negative effect on the metabolism of microbial biocatalysts. Alcohols (catechol, syringol, etc.) have been shown to act by permeabilizing the cell membrane and toxicity correlated well with the hydrophobicity of the molecule (Zaldivar et al. 2000 Biotechnol. Bioeng. 68:524-530). Organic acids (acetate, formate, etc.) are thought to cross the membrane in neutral form and ionize within the cytoplasm, inhibiting growth by collapsing the proton motive force (Palmqvist et al. 2000. Bioresour. Technol. 74:25-33, Zaldivar et al. 1999. Biotechnol. Bioeng. 66: 203-210). The inhibitory mechanisms of aldehydes are more complex. Aldehydes can react to form products with many cellular constituents in addition to direct physical and metabolic effects (Modig et al. 2002 Biochem. J. 363:769-776, Singh et al. 1995 Mutat. Res 337:9-17). In aggregate, these minor products from chemical pretreatments can retard cell growth and slow the fermentation of biomass-derived sugars (Horvath et al. 2001. Biotechnol. Bioeng. 75:540-549, Palmqvist et al. 2000. Bioresour. Technol. 74:17-24).
Furfural (a dehydration product of pentose sugars) is of particular importance. Furfural is a natural product of lignocellulosic decomposition. Furfural is also formed by the dehydration of pentose sugars during the depolymerization of cellulosic biomass under acidic conditions (Martinez et al. 2001 Biotechnol. Prog. 17:287-293). This compound is an important contributor to toxicity of hemicellulose syrups, and increases the toxicity of other compounds (Zaldivar et al. 1999. Biotechnol. Bioeng. 65: 24-33.). Furfural content in dilute acid hydrolysates of hemicellulose has been correlated with toxicity (Martinez et al., 2000. Biotechnol. Bioengin. 69(5): 526-536). Removal of furfural by lime addition (pH 10) rendered hydrolysates readily fermentable while re-addition of furfural restored toxicity (Martinez et al. 2001. Biotechnol Prog 17: 287-293). Furfural has also been shown to potentiate the toxicity of other compounds known to be present in acid hydrolysates of hemicellulose (Zaldivar et al. 1999. Biotechnol. Bioeng. 65: 24-33; Zaldivar et al. 1999 Biotechnol. Bioeng. 66: 203-210; Zaldivar et al. 2000 Biotechnol. Bioeng. 68:524-530). Furfural has been reported to alter DNA structure and sequence (Barciszewski et al. 1997 FEBS letters. 414:457-460, Khan et al., 1995 Cancer Lett. 89:95-99), inhibit glycolytic enzymes (Gorsich et al. 2006 Appl. Microbiol. Biotechnol. 71:339-349), and slow sugar metabolism (Hristozova et al. 2006. Enzyme Microbiol. Technol. 39:1108-1112).
Lignocellulosic biomass represents a potential feedstock for microbial conversion to renewable fuels and chemicals. Prior to fermentation, carbohydrate components (cellulose and hemicellulose) must be converted to soluble sugars using acids, enzymes, or a combination (Cheng et al. 2008 Biochem. Eng J 38:105-109; Wyman et al. 2005 Bioresour Technol. 96:2026-2032; Um et al. 2003 Appl Biochem Biotechnol 105:115-125). During steam pretreatment with mineral acids, 5-hydroxymethyl furfural (5-HMF) and furfural are produced as minor but toxic side products from the dehydration of hexose and pentose sugars, respectively (Martinez et al. 2000a Biotechnol Bioeng 69:526-536; Palmqvist and Hahn-Hagerdal 2000b Bioresour Technol 74:25-33). 5-HMF has been shown to retard growth and fermentation of ethanologenic E. coli (Zaldivar et al. 1999 Biotechnol Bioeng) and Saccharomyces cerevisiae (Almeida et al. 2008 Appl Microbiol Biotechnol 78:939-945; Palmqvist and Hahn-Hagerdal 2000a Bioresour Technol 74: 17-24; Taherzadeh et al. 2000 Appl Microbiol Biotechnol 53:701-708).
Furans can be removed from hemicellulose hydrolysates by over-liming to pH 10 at elevated temperatures (Martinez et al. 2000a Biotechnol Bioeng 69:526-536). This process requires the efficient separation of hydrolysate syrups from cellulosic fibers, specialized equipment for lime mixing, separation of syrups from insoluble calcium salts, and creates a solid waste for disposal. The development of furan-resistant biocatalysts could eliminate much of this process complexity. Several enteric bacterial genera (Klebsiella, Enterobacter, Escherichia, Citrobacter, Edwardsiella, Proteus) as well as yeasts have been shown to convert 5-HMF into 5-hydroxymethyl furfuryl alcohol, a less toxic compound (Boopathy et al. 1993 J Indus Microbiol 11:147-150; Palmqvist and Hahn-Hagerdal 2000a Bioresour Technol 74:17-24; Zaldivar et al. 1999 Biotechnol Bioeng 65:24-33). S. cerevisiae has been shown to produce multiple oxidoreductases (YGL157W, ADH6, and a mutated ADH1) that can reduce both 5-HMF and furfural to less toxic products (Almeida et al. 2008 Appl Microbiol Biotechnol 78:939-945; Almeida et al. 2009 Appl Microbiol Biotechnol 82: 625-638; Heer et al. 2009 Appl Environ Microbiol doi:10.1128/AEM.01649-9; Liu et al. 2009 Gene 446: 1-10). Increased expression of these genes was shown to be beneficial for some aspects of 5-HMF tolerance although none have been shown to increase the minimum inhibitory concentration of furfural. Gorisch et al. (2006 Appl Microbiol Biotechnol 71: 339-349) identified many gene inactivations in S. cerevisiae that increased sensitivity to furfural and 5-HMF. Over-expression of one gene, ZWF1 (glucose 6-phosphate dehydrogenase), increased tolerance to furfural.
The ability of fermenting organisms to function in the presence of these inhibitors has been researched extensively. Encapsulation of Saccharomyces cerevisiae in alginate has been shown to be protective and improve fermentation in acid hydrolysates of hemicellulose (Talebnia et al. 2006 J Biotechnol. 125:377-384.). Strains of S. cerevisiae have been previously described with improved resistance to hydrolysate inhibitors (Almeida et al. 2007. J. Chem. Technol. Biotechnol. 82:340-349, Martin et al. 2007. Bioresour. Technol. 98:1767-1773, Nilsson et al. 2005. Appl. Environ. Microbiol 71:7866-7871). Escherichia coli (Gutiérrez et al. 2006 J. Bacteriol. 121:154-164), S. cerevisiae (Almeida et al. 2008 Appl. Microbiol. Biotechnol. 78:939-945) and other microorganisms (Boopathy et al. 1993 J. Indust. Microbiol. 11:147-150) have been shown to contain enzymes that catalyze the reduction of furfural to the less toxic product, furfuryl alcohol (Zaldivar et al., 2000 Biotechnol. Bioeng. 68:524-530). In E. coli, furfural reductase activity appears to be NADPH-dependent (Gutiérrez et al. 2006. J. Bacteriol. 121:154-164). An NADPH-dependent furfural reductase was purified from E. coli although others may also be present. An NADPH-dependent enzyme capable of reducing 5-hydroxymethyl furfural (a dehydration product of hexose sugars) has been characterized in S. cerevisiae and identified as the ADH6 gene (Petersson et al. 2006. YEAST. 23:455-464).
The yqhD gene has been previously shown to encode an NADPH-dependent aldehyde oxidoreductase (Sulzenbacher et al. 2004. J. Mol. Biol. 342:489-502) that can be used for the production of propanediol (Nakamura et al. 2003. Current Opinion in Biotechnology. 14:454-459, Zhang et al. 2006 World Journal of Microbiology & Biotechnology. 22:945-952). This gene has also been shown to confer resistance to damage by reactive species of oxygen (Pérez et al. 2008. J. Biol. Chem. 283:7346-7353.). The dkgA gene has been shown to catalyze the reduction of 2,5-diketo-D-gluconic acid, a key step in the production of ascorbic acid (Habrych et al. 2002. Biotechnol. Prog. 18:257-2, Yum et al. 1999 Appl. Environ. Microbiol. 65:3341-3346). This enzyme is also thought to function in the reduction of methylglyoxal (Jeudy er al. 2006. Proteins 62:302-307, Ko et al. 2005. J. Bacteriol 187:5782-5789). The function of the yqfA gene is unknown but is proposed to be a membrane subunit of an oxidoreductase (Karp et al. 2007 Nucleic Acids Res. 35:7577-7590) which may be involved in fatty acid metabolism (McCue et al. 2001. Nucleic Acids Res. 29:774-782).
The Escherichia coli yqhC gene (b3010) is a predicted transcriptional regulator belonging to the AraC/XylS family of DNA-binding proteins. Inferences to date concerning the likely function of yqhC have been based solely on the similarity between the deduced protein sequence and members of the AraC/XylS family. yqhC is adjacent in the E. coli genome to the yqhD and dkgA genes, which are transcribed in the opposite orientation to yqhC.
The methods of the invention allow for the identification of enzymes that regulate the growth and ethanol production of microorganisms in the presence of furfural and/or 5-HMF. Accordingly, the ability to produce microorganisms that can grow and produce ethanol in the presence of furfural is extremely important for production of alternative sources of energy.